1. Field of the Invention
This invention is concerned with modified cellulose-containing separation media for separating particulate and/or molecular components from liquids containing same. The invention is particularly directed to separation media having utility in the pharmaceutical industry for use in the preparation and purification of pharmacologically useful liquids, i.e. parenteral solutions. Such solutions, after treatment with the separation media of this invention, are essentially free of non-specific pyrogenic reactivity.
2. Description of the Background Art
Separation of components contained in liquid may generally be divided into filtration of particulate components and separation of molecular components.
The filtration of fine particle size components from fluids has been accomplished by the use of various porous filter media through which the fluid is passed. To function as a filter, the media must allow the fluid, commonly water, through, while holding back the particulate. This holding back of the particulate is accomplished by virtue of the operation, within the porous media, of one or both of two distinctly different filtration mechanisms, namely (1) mechanical straining and (2) electrokinetic particle capture. In mechanical straining, a particle is removed by physical entrapment when it attempts to pass through a pore smaller than itself. In the case of electrokinetic capture mechanisms, the particle collides with a surface within the porous filter media and is retained on the surface by short range attractive forces.
With the exception of microporous polymeric membranes, the porous filter media known to the art as being suitable for the filtration of fine particle size particulate are typically comprised of fiber-fiber or fiber-particulate mixtures formed dynamically into sheet by vacuum felting from an aqueous slurry and then subsequently drying the finished sheet. In those fibrous filter media that depend upon mechanical straining to hold particulate, it is necessary that the pore size of the filter medium be smaller than the particle size of the particulate to be removed from the fluid. For removal of fine, submicronic particles by mechanical straining, the filter media should have correspondingly fine pores. Since the pore size of such a sheet is determined predominantly by the size and morphology of the materials used to form the sheet, it is necessary that one or more of the component materials be of a very small size, such as small diameter fibers.
As the size of the particulate sought to be removed by filtration decreases, especially into the submicron range, the difficulty and expense of providing suitably dimensioned fiber structures for optimum filtration by mechanical straining increases. Accordingly, there is considerable interest in the use of fine particulates such as diatomaceous earth in conjunction with the fibers.
However, for such materials, it is necessary to provide a matrix in order to present a coherent handleable structure for commerce and industry. Thus, at least one of the component materials in the sheet is a long, self-bonding fiber, to give the sheet sufficient structural integrity in both the wet "as formed" and in the final dried condition, to allow handling during processing, and suitability for the intended end use. Unrefined cellulose fibers such as wood pulp, cotton, cellulose acetate or rayon are commonly used. These fibers are typically relatively large, with commercially available diameters in the range of 6 to 60 micrometers. Wood pulp, most often used because of its low relative cost and fiber strength, has fiber diameters ranging from 15-25 micrometers, and fiber lengths of about 0.85 to about 6.5 mm.
Cellulose filter media sheets are conveniently formed by vacuum felting from an aqueous slurry of the component material. The vacuum felting is performed on a foraminous surface, normally a woven wire mesh which, in practice, may vary from 50 mesh to 200 mesh, with mesh openings ranging from 280 micrometers to 70 micrometers, respectively.
Assignee's U.S. Pat. No. 4,404,285 to Hou describes an embodiment wherein activated carbon in particulate form is compounded with a matrix of self-bonding cellulose fibers to form a composite sheet. The carbon particles are such that more than 90% of the particles are less than 50 microns in diameter. The composite sheet is useful for separating hormones from whole human serum.
Commonly assigned U.S. Pat. No. 4,488,969, and U.S. Ser. No. 401,361 to Hou et al., filed July 23, 1982, now U.S. Pat. No. 4,578,150, disclose a self-supporting cellulose fibrous matrix containing at least 5% by weight of fumed silica or fumed alumina. The formed silica and fumed alumina has an average particle size of less than 1 micron. The fibrous matrix is useful for delipidization and depyrogenation of fluids such as serum.
Charge modifiers have been employed to control the zeta potential of the sheet constituents and maximize performance in the electrokinetic capture of small charged contaminants. In practice, cationic charge modifiers are employed since most naturally-occurring contaminant surfaces are anionic at fluid pH of practical interest. Thus, a melamine-formaldehyde cationic colloid is disclosed for filter sheets in U.S. Pat. Nos. 4,007,113 and 4,007,114 to Ostreicher; inorganic cationic colloidal silica is disclosed in U.S. Pat. No. 4,305,782 to Ostreicher et al.; and polyamido polyamine epichlorohydrin cationic resin is disclosed in Canadian Pat. No. 1,119,105 to Hou et al. The entire disclosures of these patents are incorporated by reference herein. Such filters are sold by AMF, Inc. as ZETA PLUS.
Numerous techniques exist for the molecular separation of the components of a given sample for either analysis purposes or for product preparation purposes. One type of molecular separation embraces a variety of processes for effecting differential distribution of the sample components between two phases and such processes are generally referred to as chromatography. The differential distribution is achieved by an interchange between a moving phase, which can be a liquid or gas, and a stationary phase.
Chromatography is a general term applied to a wide variety of separation techniques based upon the sample interchange between a moving phase and a stationary phase. When gas is the moving phase (or "mobile phase" as referred to in chromatographic terminology), the technique is termed gas chromatography and when liquid is the mobile phase, the technique is termed liquid chromatography.
The collection of chromatographic techniques can be classified in several ways and the most fundamental is based on naming the types of phases used. Liquid adsorption chromatography is used extensively for organic and biochemical analysis but is limited because there are only a few suitable adsorbents. The distribution coefficient of adsorption often depends on total concentration and this behavior often results in incomplete separations. Gas-solid chromatography has generally suffered from the same defects as liquid adsorption chromatography. Ion exchange chromatography is a special field of liquid-solid chromatography and is specifically applicable to ionic species. Affinity chromatography is based on the attraction (affinity) of a ligand bonded to the solid stationary phase for a given component of the sample. Reverse phase chromatography relies on the hydrophobic characteristics of the stationary phase.
Assignee's Crowder, III, et al., U.S. Pat. No. 4,384,957, describes a column in which a mobile phase flows through a solid stationary phase; the stationary phase "system" is, broadly, a body of particulate immobilized in a porous matrix of cellulose fiber. This stationary phase has the advantage of both low pressure drop and low diffusion resistance, making it particularly suitable for commercial scale separations, particularly liquid separations. Baffle arrangements are unnecessary. As a result, it is possible to construct stable, high flow separation columns of high capacity and shorter run times which have good pressure response, freedom from channeling or fluid bypass, ease of regeneration to reproducible reuse, and the capacity to be shipped under ambient conditions or stored indefinitely. Additionally, the edges of the new stationary phase cooperate with the interior wall of the separation column to form a substantially fluid-tight seal therewith, thus preventing channeling near the walls. The entire disclosure of Crowder, III et al. is incorporated by reference herein.
The stationary phase of Crowder, III et al. comprises a porous matrix of cellulose fiber having particulate immobilized therein, wherein at least one of said fiber or particulate is effective for molecular separation. The porous matrix is substantially homogeneous with respect to each component thereof. Preferably, the particulate is effective for molecular separation. The molecular separation particulate is contained in the stationary phase at an amount effective to achieve the desired molecular separation. Among the fibers of Crowder, III et al. are cellulose fibers obtained from wood.
Assignee's U.S. application Ser. No. 576,448 to Hou et al., filled Feb. 2, 1984, a continuation-in-part of assignee's U.S. application Ser. No. 466,114, filed Feb. 14, 1983, discloses modified polysaccharide chromatographic separation media comprising polysaccharide covalently bonded to a synthetic polymer, the synthetic polymer comprising (1) a polymerizable compound which is capable of covalently coupling to the polysaccharide and (2) a polymerizable compound containing (i) an ionizable chemical gruop, (ii) a chemical group capable of being transformed into an ionizable chemical group, (iii) a chemical group capable of causing the covalent coupling of (2) to an affinity ligand or a biologically active molecule or (iv) a hydrophobic polymer. Cellulose is among the preferred modified polysaccharides.
Endotoxins, also known as bacterial pyrogens, are heat stable toxins present in bacterial cells. These materials are found primarily in gram-negative organisms and occur in the cell wall as a lipopolysaccharide (LPS) complex. These endotoxins are pyrogenic, i.e. a substance inducing fever, when injected into a mammalian species.
Biological solutions, especially those biological solutions which are intended for pharmaceutical application such as those administered by parenteral injection, must be essentially devoid of bacterial pyrogens or endotoxins. Accordingly, it is common practice in the pharmaceutical industry that biological fluids, particularly those biological fluids intended for parenteral use, be tested for pyrogen levels. One of the frequently used pyrogen level tests in the past was the rabbit pyrogen test, so called because the biological fluid in question was injected into a rabbit and the rabbit tested to determine whether fever was induced. However, this test proved to be quite cumbersome and time consuming and alternative methods of testing for pyrogens have been developed.
The use of Limulus Amebocyte Lysate (LAL) by the pharmaceutical industry has increased steadily over the last few years, both for in-process control of endotoxin contamination and for final release of medical devices in lieu of the USP pyrogen test. Guidelines for the LAL end-product use with medical devices and biological products are described by Randolph, W. F., "Licensing of Limulus Amebocyte Lysate. Use as an Alternative for Rabbit Pyrogen Test," Fed. Regist., 42, 57749 (1977). Draft guidelines have also been published for LAL use for other parenteral drugs, Randolph, W. F., "Human and Veterinary Drugs: Availability of Draft Guideline for Use of Limulus Amebocyte Lysate," Fed. Regist., 45, 3666-3669 (1980).
Traditionally, most LAL testing of pharmaceutical products has been done with the gel-clot method (Jorgensen, J. H. and Smith, R. F., "Rapid Detection of Contaminated Intravenous Fluids Using the Limulus In Vitro Endotoxin Assay," Appl. Micro. Biol. 26, 521-524 (1973)). A turbidimetric adaptation of the LAL test exists as well. The turbidimetric assay is quantitative over a continuous range of endotoxin concentrations and is often more sensitive than the gel-clot method. One such LAL turbidimetric adaptation exists for the Abbott MS-2 Microbiology System (Novitsky, T. J. et al., "Automated LAL Testing of Parenteral Drugs in the Abbott MS-2," Journ. of Parenteral Science and Technology, Vol. 36, No. 1, 11-16 (1982).
However, as disclosed in the Novitsky et al. article, the LAL test is subject to product-related inhibition or enhancement of the LAL test. And while Novitsky et al. is directed essentially to the development of a system for rapid characterization of parenteral drugs and for defining those factors that modify the LAL reaction in the presence of the drugs, essentially the article suggests a very real problem which exists with regard to the LAL test, namely that various non-pyrogenic substances alter the reactivity of the biological fluids being tested.
Further, applicants have now discovered that certain prior art cellulose-containing filtration media yield biological fluids which demonstrate unacceptable pyrogen levels when tested using Limulus Amebocyte Lysate. Further, the high pyrogen levels indicated by the LAL test of biological fluids which are filtrates of the cellulose-containing filtration media frequently are falsely positive, these same fluids showing only very low pyrogen levels when tested using the rabbit pyrogen test.
Accordingly, a need has continued to exist in the pharmaceutical industry for cellulose-containing separation media such as the type described in U.S. Pat. No. 4,384,957 to Crowder III et al., U.S. Ser. No. 576,448 to Hou et al., U.S. Pat. Nos. 4,007,113 and 4,007,114 to Ostreicher, U.S. Pat. No. 4,305,782 to Ostreicher et al., and Canadian Pat. No. 1,119,105 to Hou et al. discussed above, said media effective for the production and purification of biological fluids which are essentially free of non-specific pyrogenic reactivity using the LAL tests. Faced with an ever increasing demand for filtration media for use in the pharmaceutical industry, coupled with increasingly strict controls on the quality control of the finished product, coupled with the facility of pyrogen testing afforded by the LAL test, the development of cellulose-containing filtration media which produce biological fluid filtrates essentially free of non-specific pyrogenic reactivity as tested by the LAL test method are of great importance.